The long term objectives of this research are to elucidate the mechanisms of viral fusion proteins and to use this information to design anti-fusion anti-virals. Viral fusion proteins have been categorized by structural class (class I, class II) and by mode of activation to a fusogenic state (by exposure to low pH or interaction with host cell receptors). These features combine to yield a diversity of fusion mechanisms. While the means by which low pH activates fusion proteins are reasonably well understood, the ways in which receptors perform this task are poorly characterized. Evidence indicates that alpharetroviral fusion proteins are activated to reach a lipid interacting stage of fusion following binding to receptors at neutral pH; subsequent exposure to low pH appears to complete the process. Other viruses may use this novel two-step fusion paradigm. The first goal of this proposal is to elucidate molecular details of how a retrovirus receptor (Tva) activates its cognate fusion protein, EnvA, to reach a lipid interacting stage of fusion. The second goal is to determine the precise role of low pH at later stages of fusion. The third goal is to assess the generality and virological ramifications of this novel two-step fusion process. These goals will be achieved by addressing the following specific aims: 1 .test the hypothesis that binding of Tva to EnvA leads to conformational changes in the receptor binding subunit of EnvA that are relayed to the fusion subunit; 2.test a working model for sequential roles of receptor binding and low pH in forming distinct conformations of EnvA that mediate early and late stages of fusion; 3.test the hypotheses that conditions that activate retroviral Envs dictate the site of viral entry and influence the outcome of the infection. The experimental design employs techniques of virology, biochemistry, biophysics, cell biology, and structural biology. The project has a high degree of health relatedness. There has been progress in developing anti-fusion anti-virals that target viruses that fuse with cells at neutral pH (e.g. HIV). In contrast there are no potent anti-fusion anti-virals that target viruses that fuse with cells in low pH compartments. Hence it is important to develop methods to ascertain whether a virus requires low pH at any stage of entry and to identify ways to inhibit fusion of these classes of viral pathogens (e.g. influenza, West Nile, and Ebola viruses). The proposed work will aid these important efforts.